Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description of the Invention. The disclosures of these publications in their entireties, as well as those of U.S. Patents referenced herein, are hereby incorporated by reference in this application.
Disease resistance is an important objective of the genetic engineering of crop plants. Numerous fungi, bacteria, and other microbes are serious pests of common agricultural and forest crops. One method of controlling plant diseases has been to apply antimicrobial organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems, such as pollution of surrounding environment causing harm to humans and nontarget, beneficial organisms. A more recent method of control of microorganism pests in plants has been the use of biological control organisms which are typically natural competitors or inhibitors of the troublesome microorganisms. However, it is difficult to apply biological control organisms to large areas, and even more difficult to cause those living organisms to remain in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express antifungal compounds. This technology has given rise to additional concerns about eventual microbial resistance to well-known, naturally occurring antifungals, particularly in the face of heavy selection pressure which may occur in some areas. Thus, a continuing effort is underway to express naturally occurring antifungal compounds in plant cells directly by translation of a single structural gene. However, there is a limited pool of naturally occurring peptides and other compounds with which molecular biologists can work. Attention is now focused on the rational design of entirely new peptides which can function effectively in plant cell expression systems and in other uses where antifungal peptides can be useful.
A steadily increasing interest is being focused on defense peptides produced by a variety of organisms (Cornelissen and Melchers 1993). These peptides or their analogs have the potential as a new source for disease resistant genes. Most of these small, lytic, antimicrobial peptides have been placed into four chemically distinct groups: the magainins, the cecropins, the defensins, and the proline-rich peptides (Agerberth et al. 1991).
The cecropins, first isolated from the cecropia moth, but recently from many insects, range from 26-37 amino acids in length. Their structure includes two .alpha.-helical regions, one amphiphilic and one hydrophobic, joined by a hinge region (Christensen et al. 1988). The cecropins are thought to produce single-channel conductances in lipid bilayers such as in a cell membrane (Wade et al. 1990). Most of these peptides described to date also demonstrate a specificity to microorganisms. One exception is melittin, isolated from bee venom. This peptide is very lytic to both microorganisms and animal red blood cells. The specificity of melittin can be altered by inverting the .alpha.-helical regions or by producing cecropin A/melittin hybrids (Boman et al. 1989). Amino acid omission studies on melittin showed that deletions in the .alpha.-helical regions decreased hemolytic activity but deletions in the "hinge" region did not (Blondelle and Houghten 1991).
The magainins, from the skin of the African clawed frog (Xenopus laevus), are some of the smallest natural antimicrobial peptides yet discovered, ranging from 21-27 amino acids in length (Zasloff 1987 and Bevins and Zasloff 1990). These peptides form an amphipathic, single .alpha.-helix which can span a cell membrane. It is hypothesized that these molecules form ion channels in the microbial cell's membrane which the cell cannot control, eventually leading to lysis of the cell. The .alpha.-helix is essential for activity and changes in the amino acid sequence which stabilize this helical structure enhance the molecule's lytic activity against selected bacteria (Chen et al. 1988).
The magainins are of interest because of their ability to lyse bacterial and yeast cells but not animal cells (Soravia et al. 1988), suggesting good potential for use in agricultural and forest plant species. Different peptides from this group also demonstrated synergistic effects. When the peptide PGLa was combined with either magainin I or magainin II in a 1:1 molar ratio, the antimicrobial activity increased 20-50 fold. Interestingly, alone these peptides had no hemolytic effect but in combination they exhibited the ability to lyse a variety of eukaryotic cells (Bevins and Zasloff 1990). This complementation demonstrated synergistic effects which should be considered when studying combinations of these types of peptides.
Although all the peptides in the magainins group have similar amphipathic .alpha.-helical structures, small differences in their amino acid sequences result in different antimicrobial activity. One example of this difference can be discerned by comparing the reported activities of PGLa and XPF (Soravia et al. 1988). These two peptides are very similar in terms of structure. When tested against the bacteria Pseudomonas aeruginosa, PGLa was 4-5 times more active than XPF. When these two were tested against the yeast Candida albicans, however, XPF was 2-2.5 times more effective than PGLa. Similar differences could also be seen among different species of bacteria (Soravia et al. 1988). Even a three amino acid substitution in (Ala.sup.8,13,15)magainin II, which is reported to increase helical stability, caused a change in activities on bacterial strains (Chen et al. 1988). Applicants have observed that these structural changes increased magainin activity against bacteria but had the opposite effect against selected filamentous fungi. This indicated for the first time that the ratios of activity between different organisms could potentially be manipulated.
However, to design peptides with a differential in vitro activity between plant and fungal cells, an understanding of the structural differences between melittin, the cecropins, and magainins and how they effect antimicrobial activity was needed.
Thus, a need continues to exist for new antimicrobial polypeptides useful in improving disease resistance in plants.